ABSTRACT The ubiquitin-proteasome system (UPS) has emerged over the past 15 years as an exciting new druggable pathway for cancer therapy. The family of 100 deubiquitinating enzymes (DUBs) remove ubiquitin from sub- strate proteins. Recent studies suggest that pharmacologic inhibition of DUBs may provide an opportunity to therapeutically target oncoproteins in cancer with improved precision. Despite increasing interest in their func- tion and potential as therapeutic targets, there are limited reagents and analytical platforms to study DUBs. The goal of our interdisciplinary team (Sara Buhrlage, DUB biology, chemical biology; Jarrod Marto protein bi- ochemistry, chemical proteomics) is to develop novel DUB-focused activity reagents and related analytical methods. Achieving our milestones will directly support near-term (3-5 years) efforts by us and others in the field to develop selective tool compounds to interrogate basic DUB biology and further validate specific DUBs as cancer drug targets. We will pursue our objectives through two specific aims: Aim 1. To develop and characterize novel DUB activity-based probes (ABPs) for use in activity-based protein profiling (DUB-ABPP). We will develop an expanded collection of DUB-ABPs, including PTM- modified ubiquitin. We will validate each ABP to confirm their ability to selectively engage and enrich a subset of cellular DUBs. Probes which meet this milestone will be used in combination in multiplexed, mass spec- trometry-based ABPP to establish binding profiles for compounds in our DUB-targeting library of small mole- cules. Aim 2. To develop a data-independent thermal proteome profiling (TPP-DIA) chemoproteomic platform for DUB-targeting compounds. We will develop thermal proteome profiling (TPP) assays which will directly quantify the DUBome or proteome-wide binding behavior of DUB-targeting small molecules in live cells. We will first utilize our new DUB-ABPs as enrichment reagents to generate mass spectral libraries for DUBs and Ub-l binding proteins. We will use these reference spectra as the foundation for data-independent acquisition (DIA) thermal profiling chemoproteomic assays at the DUBome (TPP-DIA-DUBome) and proteome-wide (TPP- DIA-Proteome) levels. Compounds from our DUB-focused library spanning a range of selectivity will be ana- lyzed on these new chemoproteomic platforms. Successful completion of our objectives will provide the field with a comprehensive and powerful set of rea- gents and technologies to rapidly develop hits, leads, and probes for DUBs, and further credential members of this important, but understudied protein family as novel drug targets in cancer.